Metal-catalyzed oxidation of bovine neurofilaments in vitro

Neurofilaments (NF) are important determinants of the shape and size of nerve cells. The oxidation of NF, relevant to aging, neurodegenerative disorders, and axonal (Wallerian) degeneration, has not been studied. In this investigation, we have combined biochemical and ultrastructural methods to study the metal-catalyzed oxidation (MCO) of bovine NF using an ascorbate/Fe+3/O2 system. The oxidation of NF proteins was documented by increases in carbonyl content, which were time- and concentration-dependent. Polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses revealed the fragmentation of oxidized NF proteins, predominantly NF-H and NF-M. Electron microscopy (EM) showed that oxidized NF formed dense aggregates and bundles of laterally aggregated filaments. Finally, we also demonstrated that oxidized NF proteins were more susceptible to calpain proteolysis. In view of the growing evidence supporting increased oxidative stress on the nervous system in aging and the report of Cu/Zn superoxide dismutase mutation in familial motor neuron disease, oxidative injury of NF may be relevant to cell atrophy and degeneration of nerve cells and to the formation of abnormal cytoskeletal structures.